XB130 Deficiency Enhances Lipopolysaccharide-Induced Septic Response and Acute Lung Injury

www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 18 XB130 deficiency enhances lipopolysaccharide-induced septic response and acute lung injury Hiroaki Toba1, Tereza Tomankova1, Yingchun Wang1, Xiaohui Bai1, Hae-Ra Cho1, Zhehong Guan1, Oyedele A. Adeyi2, Feng Tian1, Shaf Keshavjee1,3,4, Mingyao Liu1,3,4 1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, Universal Health Network, University of Toronto, Toronto, Ontario, Canada 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 3Department of Surgery, University of Toronto, Toronto, Ontario, Canada 4Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Correspondence to: Mingyao Liu, e-mail: [email protected] Keywords: cell death, cell proliferation, inflammatory response, septic response, transgenic mouse Received: August 31, 2015 Accepted: March 08, 2016 Published: March 23, 2016 ABSTRACT XB130 is a novel oncoprotein that promotes cancer cell survival, proliferation and migration. Its physiological function in vivo is largely unknown. The objective of this study was to determine the role of XB130 in lipopolysaccharide (LPS)-induced septic responses and acute lung injury. LPS was intraperitoneally administrated to Xb130 knockout (KO) and wild type (WT) mice. There was a significant weight loss in KO mice at Day 2 and significantly higher disease scores during the 7 days of observation. The levels of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, interleukin-6 and interleukin-10 in the serum were significantly higher in KO mice at Day 2. In KO mice there were a significantly higher lung injury score, higher wet/dry lung weight ratio, more apoptotic cells and less proliferative cells in the lung. Macrophage infiltration was significantly elevated in the lung of KO mice. There was significantly increased number of p-GSK-3β positive cells in KO mice, which were mainly neutrophils and macrophages. XB130 is expressed in alveolar type I and type II cells in the lung. The expression in these cells was significantly reduced after LPS challenge. XB130 deficiency delayed the recovery from systemic septic responses, and the presence of XB130 in the alveolar epithelial cells may provide protective mechanisms by reducing cell death and promoting cell proliferation, and reducing pulmonary permeability. INTRODUCTION RET/PTC oncogenic kinase in thyroid cancer cells [6]. XB130 protein expression was not associated with the XB130 is a novel adaptor protein for signal postoperative prognosis of patients with hepatocellular transduction [1, 2]. Due to its high molecular similarity carcinoma [11]. By contrary, XB130 expression was with actin filament associated protein (AFAP), XB130 is an independent prognostic factor in human esophageal also known as actin filament associated protein 1-like 2 squamous cell carcinoma [12]. In gastric cancer, reduced (AFAP1L2) [3, 4]. XB130 regulates cell proliferation and XB130 protein expression is a prognostic biomarker for cell cycle progression, prevents cell death, and promotes shorter survival and a higher recurrence rate [13]. On the cell migration, through its interaction with Src protein other hand, in human ductal breast cancer, XB130 was tyrosine kinase, and its binding with p85α, the regulatory present in the cytoplasm of malignant cells, but not in the subunit of PI3K, subsequently activating the PI3K/Akt normal breast cells, and positive XB130 expression was an pathway [1, 3–9]. independent risk factor for overall survival and recurrence XB130 was found in colorectal cancer cells as a Src free survival [14]. XB130, thus, may play different role in family kinase target [10], and then found as a target of tumorigenesis depending upon the types of cancers. www.impactjournals.com/oncotarget 25420 Oncotarget Since most of cell biology studies on XB130 were disease scores, a marker of the severity of systemic sepsis, conducted in cancer cells, the physiological function of were significantly higher in Xb130 KO mice during the XB130 is largely unknown. Recently, there is a report 7 days of observation period (Figure 1C). that translocation of XB130 from cytoplasm to micro- LPS stimulation induced rapid significant increase filamentous structure is involved in NNK (a cigarette in serum levels of monocyte chemoattractant protein-1 smoking component) induced migration of normal (MCP-1), interleukin-6 (IL-6), tumor necrosis factor- human bronchial epithelial cells [8]. Moreover, the alpha (TNF-α), and IL-10 in both Xb130 KO and WT interaction between XB130 and another adaptor protein, mice (Figure 2A and 2B). It also rapidly and significantly Tks5, mediates EGF and serum-induced proliferation increased serum levels of IL-1ß, IL-12p70, IL-13, IL-17 and survival of human bronchial epithelial cells [15]. To and IFNγ (Table 1). At Day 2 after LPS treatment, most of determine the physiological function and tumorigenesis these cytokines returned to basal levels in WT mice, but of XB130 in vivo, XB130 knockout (KO) mice have the levels of MCP-1, TNF-α, IL-6 and IL-10 in Xb130 KO been generated. XB130 deficiency could slow down mice were significantly higher compared to those in WT the differentiation of proliferating basal cells to ciliated mice (Figure 2A and 2B). epithelial cells during the repair process in trachea [16]. XB130 also promotes bronchioalveolar stem cell and Club XB130 deficiency led to more severe lung injury cell proliferation in small airway repair and regeneration [17]. The objective of the present study is to determine the Acute lung injury is one of the severe complications role of XB130 in septic response and acute lung injury. of sepsis [19]. To determine the role of XB130 in LPS- Acute lung injury (ALI) and its severe form, acute induced lung injury, a separate group of animals was given respiratory distress syndrome (ARDS), are critical and LPS, or PBS as a negative control. Again, at Day 2 after leading cause of mortality in the Intensive Care Units. LPS treatment, Xb130 KO mice revealed a significant In United States, there were approximately 190,000 weight loss (Figure 3A). Significantly higher composite patients of ALI/ARDS each year with approximately disease scores were found both at Day 1 and Day 2 after 40% mortality [18]. ALI/ARDS can be caused by LPS challenge in KO mice (Figure 3B). H & E staining of intrapulmonary insults (such as infection, acid aspiration, lung tissue sections revealed more severe lung injury in ventilator-induced lung injury, ischemia-reperfusion, Xb130 KO mice at Day 2 (Figure 3C), with significantly and etc.), as well as by extrapulmonary insults (such as higher lung injury score (Figure 3D) and wet/dry lung sepsis, hemorrhagic shock, trauma, burn, and etc.) [19]. weight ratio (Figure 3E), indicating severe lung injury and Lipopolysaccharide (LPS) binds to Toll-like receptor 4, pulmonary edema. and induces the production of various pro-inflammatory The spleens harvested after 6 hours showed normal cytokines and development of ALI [20]. pulp and peri-arteriolar lymphoid cells in mice that Multiple intracellular signal transduction pathways received PBS. The spleens from KO and WT mice that mediates ALI/ARDS [21]. For example, PI3K/Akt, received LPS were congested characterized by expanded mitogen-activated protein kinase, c-Jun N-terminal red pulp relative to the peri-arteriolar lymphoid cells. After protein kinase and p38 activation are involved in the 48 hours the spleens of LPS-treated mice in both the KO overproduction of reactive oxygen species, inflammatory and WT group showed less congestion and appear similar response, cell death and alveolar epithelial cell injury [22]. to the 6 hour PBS spleens. At Day 2, the glomeruli and Since XB130 is an upstream signal of Src, and PI3K/Akt, tubules of the renal cortices show preserved architecture. we used Xb130 KO and wild type (WT) mice to determine The liver parenchyma was also intact with normal portal the role of XB130 in LPS-induced septic response and tracts and lobular architectures. No significant differences ALI. were noted between WT and KO groups in LPS-treated animals in the spleen, kidney and liver (Supplementary RESULTS Figure S1), suggesting the differences observed in the lung are organ-specific. Xb130 KO mice were more susceptible to At Day 2 after LPS treatment, more numbers of LPS-induced septic responses apoptotic (TUNEL+) cells in the lung were observed in Xb130 KO mice compared to WT mice (Figure 4A To examine the role of XB130 in LPS-induced and 4C). By contrast, immunohistochemistry staining for systemic septic response, WT and Xb130 KO mice were Ki-67, a marker for proliferative cells, was significantly challenged with 25 mg/kg LPS. Seven days’ survival of less in Xb130 KO mice (Figure 4B and 4D). Xb130 KO mice (60%) was lower than WT mice (90%), At Day 2 after LPS treatment, the number of even though it did not reach statistical significance neutrophils (Ly-6B.2+) in the lung tissue was higher in (p = 0.11) (Figure 1A). Xb130 KO mice revealed a Xb130 KO mice (Figure 5A), but did not reach statistical significant weight loss at Day 2 (Figure 1B). Composite significance (Figure 5C). On the other hand, the number www.impactjournals.com/oncotarget 25421 Oncotarget Figure 1: XB130 deficiency increased LPS-induced septic response in mice. (A) Kaplan-Meier survival curves of WT mice and Xb130 KO mice after LPS treatment (25 mg/kg). Difference in mortality was evaluated according to log-rank test (p = 0.114). Weight loss (B) and composite disease score (C) were recorded on different days. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout. Figure 2: XB130 deficiency enhanced LPS-induced cytokine production. (A and B) Cytokine/chemokine levels in serum were measured at 2, 6 and 48 hours after LPS treatment. *p < 0.05 and **p < 0.01 (WT vs. KO), #p < 0.05 and ##p < 0.01 (PBS groups vs.

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